Aonchotheca (Nematoda: Capillariidae) is validated as a separated genus from Capillaria by both mitochondrial and nuclear ribosomal DNA

Background The family Capillariidae is a group of thread-like nematodes of 27 genera and over 300 species that infect a great variety of hosts including humans. Among these, some taxa such as the genus Aonchotheca have remained controversial regarding their systematic status for decades. The aim of the current study was to verify Aonchotheca’s systemic status and to further determine whether it is a distinct genus from Capillaria using molecular and phylogenetic analyses. Results We sequenced the mitochondrial (mt) genome and nuclear small subunit (18S) rRNA gene of Aonchotheca putorii, a representative species of the genus, and investigated its systematic status in Trichinellida using maximum likelihood and Bayesian inference. The differences in amino acid sequences of 13 protein-coding genes were 12.69–67.35% among Aonchotheca, Capillaria, Eucoleus, and Pseudocapillaria with cox1 (12.69%) and atp8 (67.35%) as the most and the least conserved gene, respectively, and the difference of two mt rRNAs was 18.61–34.15%. Phylogenetic analyses of the complete mt genome and 18S rRNAs unequivocally showed that Aonchotheca was a distinct genus from Capillaria. Conclusions Large difference exists among Aonchotheca, Capillaria, Eucoleus, and Pseudocapillarias. Aonchotheca putorii is the first species in the genus Aonchotheca for which a complete mitogenome has been sequenced. These data are useful for phylogenetics, systematics and the evolution of Capillariidae. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-022-05609-9.

liver, severely damaging them [1,3]. However, the identification of Capillariidae to species or even genus by morphology alone is one of the most complicated and challenging tasks among all nematodes. Consequently, controversies on some taxon remain until now [4,6,7]. Some taxonomic experts have proposed several new genera and synonymies of capillariids and suggested reclassifying this group [1,3].
The use of DNA-based molecular techniques is one of the recent advancements for the accurate identification and understanding of nematode evolutionary relationships [8,9]. The nuclear small subunit of the ribosomal gene (18S rRNA) has been frequently used to analyze phylogenetic relationships among nematodes at different taxonomic levels, especially of some representative capillariids [5,[10][11][12][13][14]. Genome-based phylogenetic analyses such as mitochondrial (mt) genome (mitogenome) are increasingly utilized by the taxonomist for a better understanding of nematodes' relationships at various taxonomic levels and/or judgment of the discordances and congruences with morphology-based and nuclear rDNAbased phylogenies due to the shortcomings of 18S rRNA [15,16]. Within the family Capillariidae, the currently available mitogenomes are representatives of the genus Capillaria Zeder, 1800 (MH665363), Eucoleus Dujardin, 1845 (NC_056391), and Pseudocapillaria Freitas, 1959 (MZ708958).
Members of the genus Aonchotheca López-Neyra, 1947, are prevalent in many geographical regions and mainly infect the stomach and intestines of wild carnivores, especially mustelids [17]. They utilize earthworms as a reservoir to enhance their infection to the definitive hosts [18]. The taxonomic status of Aonchotheca putorii (Rudolphi, 1819) López-Neyra, 1947, within Capillariidae has also been revised several times, and it is often considered a species of the genus Capillaria [18]. Intraspecific morphometric variations among capillariids have been frequently observed because of their phenotypic plasticity, physiological and/or immunological effects of different hosts or even processing techniques in different laboratories [19]. Tamaru et al. observed significant nucleotide variation in the 18S rRNA sequence among specimens of A. putorii collected from various wild carnivores in Japan [19]. In contrast, Kołodziej-Sobocinska et al. did not observe significant genetic divergence in it among A. putorii worms collected from either same or different hosts at distant geographical localizations [20]. Hence, the two aims of the current study were to (1) analyze the genetic divergence in the 18S rRNA sequence of A. putorii from different hosts and locations and (2) verify the status of Aonchotheca by comparing mt coding regions and analyzing phylogenetic relationships within the family Capillariidae.

Parasites and molecular identification
Adult parasites were collected from the small intestine of dead hedgehogs (Erinaceus europaeus Linnaeus, 1758) originated from Beijing, China. The specimens were intensively washed in ultrapure water and physiological saline solution, and then stored in 75% ethanol. They were morphologically identified as capillariid nematodes with the presence of precloacal caudal alae in male worms [5]. For molecular identification, the total genomic DNA was extracted from several worms using QIAamp ® DNA Micro Kit (Qiagen, Hilden, Germany) per the manufacturer's instruction. Polymerase chain reaction (PCR) was applied to amplify mitochondrial (mt) gene cox1 using the primers JB3-JB4.5 as previously described [21] and nuclear 18S rRNA by the newly designed ones (F: 5ʹ-TTG GTG CGT TCG GTT CGC TGT T-3ʹ; R: 5ʹ-CCA AGC GAG CAG CAT CAG TCC A-3ʹ). PCR was carried out in BIO-RAD T100 ™ thermal cycler (Bio-Rad, Hercules, CA, USA) using the following conditions: 95 ℃ 1 min, followed by 37 cycles of 98 ℃ 10 s, 60 ℃ 30 s and 72 ℃ 1 min, with a final extension at 72 ℃ 8 min. The PCR amplicons were purified using NucleoSpin ® Gel and PCR Clean-up (Takara Bio USA, Inc.) and sent to BGI Co., Ltd. (Shenzhen, China) for direct sequencing in both directions. The DNA sequences were assembled by DNAMAN v6.0 (Lynnon Biosoft, USA). These sequences were then used to BLAST search GenBank to confirm their identities and species identification of the worms of DNA origin.

Sequencing and assembling mitochondrial genome
Total genomic DNA was fragmented to about 350 bp. The DNA libraries were sequenced using Illumina Hiseq 6000 platform (Novogene Co. Ltd. Tianjin, China). The raw sequences were in FASTQ format. Clean DNA sequences were obtained by removing adapter sequences, low-quality bases (Phred quality < 5), or uncertain reads with repetitive "N" bases. The partial cox1 sequence derived from PCR amplicons as described earlier was then used as the initial reference to assemble the complete mitogenome sequence of A. putorii using Geneious Prime v.2022.0.1 [22] with the following parameters: (i) minimum overlap within the range of 150-200 bp; (ii) minimum overlap identity of 99%; (iii) maximum gap of 5 bp. The complete circular mitogenome of the roundworm was then verified by PCR with primers as listed (Additional files 1 and 2: Table S1 and Figure S1).

Annotation of mitochondrial genome
The webserver MITOs was used for the preliminary annotation of 37 genes in the above-assembled mitogenome [23]. Initiation/termination codons and gene boundaries for all 13 protein-coding genes (PCGs) were identified by Open Reading Frame (ORF) Finder of NCBI (https:// www. ncbi. nlm. nih. gov/ orffi nder/). Locations and borders of two rRNAs (rrnL and rrnS) were recognized based on Tandem [24] and aligned capillariid sequences. Similarity, sequences, directions, and the secondary structures of 22 transfer RNA genes (tRNAs) were further verified by tRNAscan-SE 2.0 with a cutoff score of 1.0 [25]. MEGA v11.0 was used to verify accuracies of 13 PCGs and infer 13 amino acid sequences, codon usage and relative synonymous codon usage (RSCU) [26]. The values of the non-synonymous (Ka)/ synonymous (Ks) substitution ratios were calculated with DnaSP v5 to understand the selective stress in the evolution of the family Capillariidae.

Phylogenetic analyses
The 18 complete mitogenomes in the order Trichinellida available in GenBank were all included in phylogenetic analyses. To better understand the evolution and phylogenetic relationships of Trichinellida lineage, we used Xiphinema pachtaicum Tulaganov, 1938 (GenBank no. NC_033870) and X. rivesi Dalmaso, 1969 (GenBank no. NC_033869) as outgroups [27] (Table 1), as Dorylaimida evolves more rapidly in their mitogenome than Trichinellida within the class Enoplea [10]. The aligned amino acid sequences by MAFFT online server with "L-INS-I" [28] were concatenated into a single alignment database and uploaded to Gbolcks 0.91b [29] to exclude ambiguity and identify more conserved blocks by selecting the option for "more stringent. " Bayesian inference (BI) and maximum likelihood (ML) were applied to phylogenetic analyses. Using Mrbayes 3.2 [30] with default model "JC69, " BI tree was constructed with four independent Markov chains by analyzing 1,000,000 generations and sampling tree every 100 generations. Based on ModelFinder in IQTree v.2.1.3 and ProtTest 3.4.2 [31,32], "mtZOA + F + R5" and "MtArt + I + G + F" were selected as the most suitable models for ML analysis in IQTree v.2.1.3 and PhyML 3.1, respectively. Additionally, available 18S rRNA sequences within the family Capillariidae were used to analyze nuclear phylogenetic relationships and genetic distances. The 18S rRNA sequences were aligned to a single database and used to constructed phylogenetic analyses based on ML (best model: GTR + I + G) and BI (best model: GTR) with the same steps as above. The alignment database was also applied to generate genetic distances of 18S rRNA using MEGA v11.0 [26].

First molecular identification of A. putorii in China
The mt cox1 sequence of PCR amplicon of 652 bp (GenBank accession no: OP363931) using primer pair JB3-JB4.5 as previously described [21] showed only 81.7% identities to GenBank no. MH665361 by BLAST search, which was a Capillaria sp. This low identity indicates the parasitic worm may be one member of the family Capillariidae and relate to Capillaria sp. species. We then PCR amplified and sequenced 18S rRNA of 1813 bp. The newly obtained 18S rRNA sequence (GenBank accession no: OP028951) of this nematode showed 100% identity to GenBank no. LC052349 of A. putorii. Therefore, the nematodes recovered from the small intestine of hedgehogs in Beijing, China, are identified as A. putorii, which is the first report in the country to our knowledge. Aonchotheca. putorii has been found in a wide range of geographical locations and several mammalian hosts including minks, weasels, martens, racoons, and even the domestic cats [17].

First complete mt genome of A. putorii
We then tried to decode the whole mt genome of A. putorii. Due to the lack of mt genome of closely related Twenty-five intergenic regions ranging from 1 to 72 bp in length were found scattered throughout the mitogenome. The highest AT content of 84.8% was in intergenic regions, and AT-skew in these regions was positive while the value of GC-skew in them was "0" (the number of G bases was roughly equal to C bases, Table 3). The mitogenomes of nematodes usually contain two noncoding regions (NCRs) with different sizes [15,33,34]. The length of AT-rich was 137 bp in Necator americanus Stiles, 1902 [35], 886 bp in Ascaris suum Goeze, 1782 [36], and approximately 7 kb in Hoplolaimus columbus Sher, 1963 [37]. As far as members of the family Capillariidae were concerned, no AT-rich region was found in the mitogenome of A. putorii, but it was 306 bp, 121 bp and 99 bp in E. annulatus [33], Capillaria sp. (MH665363) and P. tomentosa (MZ708958), respectively.

Characteristics of protein-coding genes
Aligning with available sequences of capillariid nematodes (GenBank no. MZ708958, NC_056391 and MH665363), the boundaries of each PCG were determined, and the initiation/termination codons and the directions of translation were identified. For A. putorii, the longest PCG was cox1 (1584 bp), followed by nad5 (1521 bp). The rest were nad4 (1,263 bp), cytb (1136 bp), nad1 (912 bp), atp6 (909 bp), nad2 (900 bp), cox3 ATN was exclusively used as an initiation codon in all PCGs where N was T, A or G. ATT was the most frequent initiation codon used in six PCGs (nad2, nad6, atp8, nad3, cox1 and nad1), followed by ATA in five (for  nad5, nad4, nad4L, atp6 and cox2). The least favored start codon was ATG used only in cytb and cox3. The complete termination codon TAA was the most common one, which was used in all PCGs except cytb, atp8, and nad3, in which incomplete stop codons TA and T were used, respectively. Start codons ATT, ATA and ATG were shared among those species, while TTG was only used in E. annulatus and Capillaria sp., and ATC was unique in P. tomentosa. Complete termination codon TAA was prevalent among those species, but TAG was used in E. annulatus, Capillaria sp. and P. tomentosa excluding A. putorii.
The GC-and AT-skews for mitogenome are calculated as a measure of the compositional asymmetry and effective assistant of replication [38,39]. For the A. putorii mitogenome, the AT-and GC-skews were − 0.082 and − 0.157, respectively, which showed no clear trend of A or T bases, but a significant use of C bases compared with other nematodes [40]. Similar bases trend also showed in PCGs. AT and GC-skews of 13 PCGs were from − 0.349 (nad6) to 0.071 (nad4L) and from − 0.345 (nad2) to 0.345 (cytb) ( Table 3), indicating a trend of A bases among those PCGs that would have an impact on RSCU. RSCU represents an intuitive reflection of the use of codon bias, and a higher RSCU indicates a higher codon usage bias. Within codons encoding amino acids, there was an obvious bias of T-or A-rich codons. For example, Leu was encoded by TTA, TTG, CTT, CTC, CTA and CTG, but the RSCU value of TTA was 3.91 higher than that with C and G residues (Table 4). In PCGs, Ka/Ks test was helpful for better understanding the selective constraints, evolutionary changes, sexual selection and disease resistance [41]. Generally, the ratio of Ka/Ks being > 1 represents rapid evolution of a protein gene and a positive selection and changes in protein's functions. In contrast, < 1 indicates a negative selection and selectively purified among genes [42,43]. In the present study, the Ka/Ks ratios of the genes atp6, cox3, nad3, nad4 and nad6 ranged from 1.07 to 1.72 (Fig. 2), indicating these genes had experienced a positive selection or had undergone functional changes. The Ka/Ks values of atp8, cox1, cox2, cytb, nad1, nad2, nad4L and nad5 were < 1 (Fig. 2), suggesting they were highly constrained within the family Capillariidae. Consistent with previous reports, the findings also indicated that cox1 was selectively purified and might play an adaptive role in the evolutionary process of Capillariidae species, and it can be used as useful marker to identify and distinguish capillariid species. Interestingly, atp8 gene was not only found in those species, but under purifying selection across Capillariidae, though it was variable protein-gene or even missing in some nematodes [44].

Transfer and ribosomal RNA genes
All 22 tRNAs were obtained in the present study ranging from 54 to 71 bp in length. The predicted secondary structures of most tRNAs identified in this study were similar to those found in other nematodes, and the TΨC stem loop and variable loop were replaced by a TVreplacement loop structure, which was common in the mitogenomes of nematodes excluding Trichinella spiralis (Owen, 1835) Railliet, 1895 [44][45][46]. Consistent with previous reports, the predicted structure of tRNA-Ser (AGN and UCN) lacked the DHU-arm, which was replaced by 4-5 nucleotide residues [47,48]. In the present study, 9 of 20 tRNAs (tRNA-His, tRNA-Ile, tRNA-Arg, tRNA-Lys, tRNA-Leu CUN , tRNA-Leu UUR , tRNA-Met, tRNA-Trp, and tRNA-Tyr) presented relatively standard "cloverleaf " secondary structures like those of T. spiralis [44] (Fig. 3). The length of nine TΨC stems was from 2 to 5 bp, and the length of variable arms between the anticodon loop and TΨC stem-loop ranged from 2 to 8 bp.
The locations and boundaries of rrnL (16S rRNA) and rrnS (12S rRNA) were identified by alignment with available Capillariidae species (NC_056391). The typical arrangement of the rrnL was located between tRNA-Val and atp6, and the rrnS was situated in tRNA-Ser AGN and tRNA-Val, similar to Capillaria sp. (MH665363), E. annulatus (MW999680) [33] and P. tomentosa (MZ708958). The length of these two rRNAs was 864 bp and 693 bp, respectively ( Table 2).

Comparative analyses of mitochondrial sequence and nuclear 18S rRNA
Comparisons of nucleotide sequences of mt genes are listed in Table 5 (Table 5). Furthermore, cox1 and atp8 were the most and the least conserved PCG, respectively. The differences of rRNAs nucleotide sequences were 18.61-30.99% in rrnL and 22.03-34.15% in rrnS (Table 5). It is acceptable that divergence Fig. 3 The secondary structures of 22 tRNA of Aonchotheca putorii mitochondrial genome. Eleven tRNAs have a typical TV-loop and nine tRNAs relatively standard "cloverleaf" structures; Leu1 and Leu2 for codon families CUN and UUR, respectively; S1 and S2 for codon families UCN and AGN, respectively of mt DNA sequences between species in nematodes was 10-20% [49]. The differences observed in the current study in nucleotide sequences of all 13 PCG and two rRNA genes ranged from 17.57 to 48.23%, and the divergences in amino acids were from 12.69 to 67.35% clearly indicated A. putorii was a distinct species from other Capillariidae species. The differences among Aonchotheca, Capillaria, Eucoleus, and Pseudocapillaria were higher than species level and further suggested Aonchotheca was a distinct genus.

Phylogenetic analyses
BI and ML trees of 18S rRNAs of Capillariidae species (Additional file 2: Figure S1) showed similar topologies to previous studies [5,50]. These indicated some Capillaria species did not belong to this genus. The tree further proved that genera Capillaria and Eucoleus were all monophyly and more related, the genus Aonchotheca might be paraphyly, and results also showed a closer relationship among genera Aonchotheca, Calodium, Pearsonema, Pseudocapillaria, and Baruscapillaria with moderate supports (Additional file 3: Figure S2).
Using X. pachtaicum and X. rivesi as the outgroups in analyzing phylogenetic relationships within class Trichinellida, both BI and ML trees displayed similar topological and systematic relationships (Fig. 4), which was consistent with previous data [33,51]. The families Capillariidae, Trichinellidae and Trichuridae were clearly in their own clades. In accord with previous studies based on complete mitogenome or 18S rRNA sequences [33,50,51], our results also showed those families were monophyletic with Capillariidae and Trichuridae forming sister taxa of high statistical support (Bpp = 1, Bf ≥ 98).
In the family Capillariidae, Capillaria was a sister taxon genus to Eucoleus, Pseudocapillaria and Aonchotheca, of high support (Bpp = 1, Bf ≥ 99). Furthermore, there was a closer relationship between genera Aonchotheca and Pseudocapillaria, and between Capillaria and Eucoleus, which was similar to the previous results by analyzing cox1 and 18S rRNA [4,5,50,51], indicating cox1 and 18S rRNA genes were useful markers to mark capillariids. In addition, the distances between the Capillaria species (Capillaria sp.) and the other three capillariid nematodes (E. annulates, P. tomentosa and A. putorii) were longer than the distances within other trichinellid species, which further clarified long genetic distance among those genera. These phylogenetic results also indicated that Eucoleus, Pseudocapillaria, and Aonchotheca were three distinct genera from Capillaria, supporting the accuracy of Moravec taxonomic revision [3,5,50]. However, there are still taxonomic controversies within Capillariidae concerning phylogenomics and

Conclusion
We have revealed details of the mitogenome of A. putorii and showed that Aonchotheca is a distinct genus from Capillaria. Our data indicate mitogenome is an ideal tool for analyzing the phylogenetic relationships and dealing with systematic controversy. In addition, nuclear DNA of 18S rRNA agrees with the phylogenetic results of mitogenome. Though the molecular information in capillariids has steadily increased recently, mt DNA sequences are still in great demand to reveal evolutionary rate, phylogenomic and systematic relationships within this family.
Additional file 1: Table S1. The designed primers were used to amplify the complete mitochondrial sequence of Aonchotheca putorii. Table S2 The uncorrected paired genetic distance of 18S rRNAs among the Capillariidae species. Table S3 The uncorrected paired genetic distance of 18S rRNAs among the Aonchotheca putorii from different regions.
Additional file 3. Figure S2. The phylogenetic analyses based on 18S rRNA among the family Capillariidae using Bayesian posterior probability